Induction of mammary gland development in estrogen receptor-alpha knockout mice

Mammary glands from the estrogen receptor-a knockout (alphaERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERalpha signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the alphaERKO mammary phenotype. We report that circulating PRL is reduced in the female alphaERKO mouse. Implantation of an age-matched, heterozygous ERalpha pituitary isograft under the renal capsule of 25-day-old or 12-week-old alphaERKO mice increased circulating PRL and progesterone levels, and induced mammary gland development. Grafted alphaERKO mice also possessed hypertrophied corpora lutea demonstrating that PRL is luteotropic in the alphaERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in alphaERKO mice despite elevated PRL levels. Hormone replacement using pellet implants demonstrated that pharmacological doses of estradiol induced limited mammary ductal elongation, and estradiol in combination with progesterone stimulated lobuloalveolar development. PRL alone or in combination with progesterone or estradiol did not induce alphaERKO mammary growth. Estradiol and progesterone are required for the structural development of the alphaERKO mammary gland, and PRL contributes to this development by inducing ovarian progesterone levels. Therefore, the manifestation of the alphaERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen signaling at the hypothalamic/pituitary axis.     

Estrogen receptor alpha and imprinting of the neonatal mouse ventral prostate by estrogen

Exposure to estrogen in the neonatal period affects prostatic growth and leads to an increased incidence of prostatic intraepithelial neoplasia in later life. The effects of neonatal estrogen are clearly dependent on estrogen receptor (ER) alpha because they do not occur in ERalpha-knockout mice. Because ERalpha is expressed in the stroma, but not in the epithelium, of the adult ventral prostate, the concept of indirect estrogen effects through stromal signaling has been proposed. Here, we show that during the first 4 weeks of life, there are profound and rapid changes in the ER profile in the mouse ventral prostate. ERalpha is abundant in the stroma during week 1, but by week 2 it is exclusively epithelial, and then by week 4, ERalpha is lost and ERbeta is dominant in the prostatic epithelium. The presence of ERalpha is associated with a high proliferation index, and ERbeta is associated with quiescence. Branching morphogenesis was altered in ERalpha-/-, but not in ERbeta-/-, mice. We conclude that imprinting and branching morphogenesis of the ventral prostate are mediated by estrogen acting directly on epithelial and stromal ERalpha during the first 2 weeks of life.     

Classical estrogen receptor alpha signaling mediates negative and positive feedback on gonadotropin-releasing hormone neuron firing

During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.     

In vivo inhibition of keratinocyte growth factor receptor expression by estrogen and antagonism by progesterone in the mouse mammary gland.

Mammary gland development is regulated by complex interactions among mammogenic hormones and locally derived paracrine growth factors. In epithelial tissues, keratinocyte growth factor (KGF or FGF-7) originates in the stroma while its receptor (KGFR or FGFR2-IIIb) is present only in the epithelium. Previous work showed that estrogen but not progesterone could stimulate the synthesis of KGF in mammary stroma in vivo. The effects of 17 beta-estradiol and progesterone on KGFR expression in vivo were examined in these studies. Peripubertal and mature virgin mice received subcutaneous injections of hormone in sesame oil after which KGFR mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. Estradiol treatment caused a dose- and time-dependent decrease in KGFR mRNA level in mice from both age groups while stimulating ductal growth after 7 days of treatment. Inhibition of KGFR expression was near maximal at an estradiol dose of 2 microg after 1 day of treatment. Progesterone injection increased KGFR mRNA levels but this effect correlated with the stimulation of ductal growth. However, when progesterone was co-administered with estradiol, KGFR mRNA levels were maintained in the absence of any effect on ductal growth. Thus, estradiol inhibited KGFR mRNA only when elevated unopposed by progesterone. These data show that KGFR expression is determined by the ratio of estradiol and progesterone and suggests a mechanism through which these hormones can co-operate to optimize their growth-promoting effects. Consequences of hormone imbalance are also implicated.     

Expression of BRCA1 gene in ewe mammary epithelial cells during pregnancy: regulation by growth hormone and steroid hormones.

OBJECTIVE: Steroid hormones (estradiol and progesterone) in association with prolactin and growth hormone are involved in lobulo alveolar development of the mammary gland during pregnancy. We hypothesized that the BRCA1 gene may be induced by these different hormones. METHODS AND RESULTS: In this study, we have demonstrated by Northern blot and in situ hybridization, that the expression of ovine (o) BRCA1 mRNA in mammary epithelial cells increased dramatically during a short period in the second half of pregnancy (days 70 to 112) and decreased at the end of pregnancy. The increase in oBRCA1 mRNA expression is concomitant with rapid lobulo alveolar growth. Using an in vivo protocol to artificially induce mammary gland development, we demonstrated by the real-time RT-PCR method that growth hormone in association with estrogen, progesterone and hydrocortisone induces an increase of BRCA1 mRNA expression in the ewe mammary gland. Moreover, we showed that estradiol and progesterone induce oBRCA1 expression in primary cultures of ewe mammary gland. CONCLUSIONS: These results suggest that BRCA1 is a potential regulator of the effects of steroid hormones and growth hormone in the induction of mammary epithelial cell proliferation.     

Estrogen receptors alpha and beta in the rodent mammary gland

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERalpha and ERbeta, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERalpha was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERalpha with up to 70% of the nuclei positive at day 21. Approximately 60-70% of epithelial cells expressed ERbeta at all stages of breast development. Cells coexpressing ERalpha and ERbeta were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERalpha-positive cells but was observed in 3-7% of ERbeta-containing cells. Because more than 90% of ERbeta-bearing cells do not proliferate, and 55-70% of the dividing cells have neither ERalpha nor ERbeta, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.     

Prolactin regulates mammary epithelial cell proliferation via autocrine/paracrine mechanism

Prolactin (PRL) is essential for a number of developmental events in the mammary gland. Work with PRL and PRL receptor knockout mice has shown that PRL indirectly regulates ductal side branching during puberty and directly controls lobuloalveolar development and lactogenesis during pregnancy. Anterior pituitary or placental PRL is thought to be responsible for these functions via an endocrine mechanism; however, PRL is also produced in a number of extrapituitary sites including the mammary gland. The physiologic relevance of mammary PRL remains unknown. In this study we utilized mammary recombination in Rag1^−/− hosts, to determine whether mammary PRL plays a role in the regulation of mammary gland development. Mammary glands formed with the PRL gene deleted from either the epithelium, stroma, or both displayed normal development, on the basis of whole mount and hematoxylin and eosin histology, during puberty and lactation. At the end of pregnancy, a 2.8-fold decrease in bromodeoxyuridine incorporation was observed in the epithelial cells of mammary glands formed using PRL knockout epithelium compared with those formed using wildtype epithelium. No balancing alteration in the rates of apoptosis was detected. Thus, mammary-derived PRL influences mammary epithelial cell proliferation via an autocrine/paracrine mechanism, establishing a physiologic function for mammary PRL during mammopoiesis.     

Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy

Full term pregnancy early in life is the most effective natural protection against breast cancer in women. Rats treated with chemical carcinogen are similarly protected by a previous pregnancy from mammary carcinogenesis. Proliferation and differentiation of the mammary gland does not explain this phenomenon, as shown by the relative ineffectiveness of perphenazine, a potent mitogenic and differentiating agent. Here, we show that short term treatment of nulliparous rats with pregnancy levels of estradiol 17beta and progesterone has high efficacy in protecting them from chemical carcinogen induced mammary cancers. Because the mammary gland is exposed to the highest physiological concentrations of estradiol and progesterone during full term pregnancy, it is these elevated levels of hormones that likely induce protection from mammary cancer. Thus, it appears possible to mimic the protective effects of pregnancy against breast cancer in nulliparous rats by short term specific hormonal intervention.     

Ontogeny and control of prolactin receptors in the mammary gland and liver of virgin, pregnant and lactating rats.

Prolactin receptors were identified and partially characterized in the mammary gland of the rat. The binding of 125I-labelled ovine prolactin to a subcellular particulate fraction of rat mammary gland decreased between days 30 and 100 of age. Over the same period, binding to the liver increased and there was a significant negative correlation between prolactin binding in the two tissues. Binding to the mammary gland was low during pregnancy, increased in early lactation and declined after the litters were weaned. Binding to the liver was lower during lactation than during pregnancy or the period after weaning suggesting that tissue-specific factors may operate in the control of this receptor. In virgin rats, prolactin binding by the mammary gland was increased by oestrogen. This effect was blocked by hypophysectomy and partially restored by replacement therapy with prolactin. Hypothyroidism and treatment with progesterone also reduced the response to oestrogen. The maintenance of prolactin binding by the mammary gland of lactating rats depends on the presence of the ovaries and pituitary, thyroid and adrenal glands. Examination of the ratio epithelium: stroma suggests that prolactin acts by increasing the number of epithelial cells in the mammary gland and that thyroid, adrenal and ovarian hormones modulate the number of receptors per cell.     

Early exposure of the rat mammary gland to estrogen and progesterone blocks co-localization of estrogen receptor expression and proliferation

An early single full-term pregnancy induces a long-lasting protective effect against mammary tumor development in humans and rodents. This protective effect can be mimicked in rats by short-term administration of estrogen and progesterone hormones prior to carcinogen administration. The hormones of pregnancy are able to induce a proliferative block upon carcinogen challenge that is not observed in the age-matched virgin. We wished to determine whether carcinogen is needed to induce a paracrine-to-autocrine shift of proliferation in steroid receptor positive cells or if such a cell population already exists in the age-matched virgin mammary gland. Here we show that estrogen receptor positive (ER+) proliferating cells are rare in the developing mammary gland of the virgin rat but represent the majority of the proliferating cells in the mature (96-day-old) mammary gland of the virgin rat. As the majority of the proliferating cells before carcinogen challenge were ER positive, the ER+ proliferating cells in the mature mammary gland may represent the target cells for carcinogen-induced transformation. Importantly, prior exposure of the mammary gland to pregnancy levels of estrogen/progesterone blocked this positive association. This ability to block the proliferation of the ER+ cells may be one factor by which pregnancy induces protection against breast cancer.     

Quantitative analysis of estrogen receptor proteins in rat mammary gland.

Estrogen receptor alpha and beta proteins (ERalpha and ERbeta) at various stages of development of the rat mammary gland were quantified by Western blotting. ERalpha and ERbeta recombinant proteins were used as standards, and their molar concentrations were measured by ligand binding assays. In 3-week-old pregnant, lactating, and postlactating rats the ERalpha content ranged from 0.30-1.55 fmol/microg total protein (mean values). The ERbeta content of the same samples ranged between 1.06-7.50 fmol/microg total protein. At every developmental stage, the ERbeta content of the mammary gland was higher than that of ERalpha. When receptor levels were normalized against beta-actin, it was evident that ER expression changed during development, with maximum expression of both receptors during the lactation period. With an antibody raised against the 18-amino acid insert of the ERbeta variant, originally called ERbeta2 but named ERbetains in this paper, Western blots revealed that ERbetains protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERbetains paralleled those of the protein. Double immunohistochemical staining with anti-ERalpha and anti-ERbetains antibodies revealed that ERbetains protein colocalized with ERalpha in 70-80% of the ERalpha-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERbetains is regulated not only quantitatively, but also with regard to its cellular distribution. As ERbetains acts as the dominant repressor of ERalpha, we suggest that its coexpression with ERalpha quenches ERalpha function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation.     

Role of estrogen receptor beta in uterine stroma and epithelium: Insights from estrogen receptor beta-/- mice

In this study, we compared the uterine tissue of estrogen receptor (ER)beta(-/-) mice and their WT littermates for differences in morphology, proliferation [the percentage of labeled cells 2 h after BrdUrd injection and EGF receptor (EGFR) expression], and differentiation (expression of progesterone receptor, E-cadherin, and cytokeratins). In ovariectomized mice, progesterone receptor expression in the uterine epithelium was similar in WT and ERbeta(-/-) mice, but E-cadherin and cytokeratin 18 expression was lower in ERbeta(-/-) mice. The percentage of cells in S phase was 1.5% in WT mice and 8% in ERbeta(-/-) mice. Sixteen hours after injection of 17beta-estradiol (E(2)), the number of BrdUrd-labeled cells increased 20-fold in WT mice and 80-fold in ERbeta(-/-) mice. Although ERalpha was abundant in intact mice, after ovariectomy, ERalpha could not be detected in the luminal epithelium of either WT or ERbeta(-/-) mice. In both untreated and E(2)-treated mice, ERalpha and ERbeta were colocalized in the nuclei of many stromal and glandular epithelial cells. However, upon E(2) + progesterone treatment, ERalpha and ERbeta were not coexpressed in any cells. In WT mice, EGFR was located on the membranes and in the cytoplasm of luminal epithelium, but not in the stroma. In ERbeta(-/-) mice, there was a marked expression of EGFR in the nuclei of epithelial and stromal cells. Upon E(2) treatment, EGFR on cell membranes was down-regulated in WT but not in ERbeta(-/-) mice. These findings reveal an important role for ERbeta in response to E(2) and in the organization, growth, and differentiation of the uterine epithelium.     

Mammary gland development in adult mice requires epithelial and stromal estrogen receptor alpha

Complete mammary gland development takes place following puberty and depends on the estrogen receptor (ER)alpha and the progesterone receptor (PR) and is tightly regulated by the interaction of the mammary epithelium with the stromal compartment. Studies using mammary tissues of immature mice have indicated that stromal but not epithelial ER alpha is required for mammary gland growth. This study investigates whether these same tissue growth requirements of neonate tissue are necessary for mammary development and response in adult mice. Mammary epithelial cells were isolated from adult mice with a targeted disruption of the ER alpha gene (alpha ERKO) or from wild-type counterparts and injected into epithelial-free mammary fat pads of 3-wk-old female alpha ERKO or wild-type mice. Ten weeks after cell injection, analysis of mammary gland whole mounts showed that both stromal and epithelial ER alpha were required for complete mammary gland development in adult mice. However, when the mice were treated with high doses of estradiol (E2) and progesterone, stromal ER alpha was sufficient to generate full mammary gland growth. Surprisingly, ER alpha-deficient epithelial cells were able to proliferate and develop into a rudimentary mammary ductal structure in an ER alpha-negative stroma, indicating that neither stromal nor epithelial ER alpha are required for the mammary rudiment to form in the adult mouse, as confirmed by the phenotype of the alpha ERKO mammary gland. Use of this in vivo model system has demonstrated that neonatal and adult mammary tissues use a different tissue-specific role for ER alpha in mammary response. Immunostaining for ER alpha and PR in the mammary outgrowths supported the view that both stromal and epithelial ER alpha, in cooperation with epithelial PR, govern mammary gland development in adult mice.